WO1999032607A1 - Materiel biologique derivant de membranes basale de follicules - Google Patents

Materiel biologique derivant de membranes basale de follicules Download PDF

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Publication number
WO1999032607A1
WO1999032607A1 PCT/US1998/027289 US9827289W WO9932607A1 WO 1999032607 A1 WO1999032607 A1 WO 1999032607A1 US 9827289 W US9827289 W US 9827289W WO 9932607 A1 WO9932607 A1 WO 9932607A1
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cells
basal lamina
fraction
basement membrane
granulosa
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PCT/US1998/027289
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English (en)
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Elikplimi K. Asem
John J. Turek
J. Paul Robinson
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Purdue Research Foundation
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Priority to AU20907/99A priority Critical patent/AU2090799A/en
Priority to EP98965443A priority patent/EP1042453A4/fr
Priority to US09/582,179 priority patent/US6485969B1/en
Publication of WO1999032607A1 publication Critical patent/WO1999032607A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to a basement membrane composition and method for its preparation and use. More particularly, the present invention is directed to the basement membrane of avian and reptile follicles and the use of same to support the growth and differentiation of eukaryotic cells cultured in vitro.
  • Basement membranes also known as basal lamina
  • ECMs extracellular matrices
  • basement membranes influence several physiological and pathological processes. Accordingly, complete and intact basement membrane is naturally functions as a substrate for cell proliferation and growth. However, intact basement membranes are not commercially available for research.
  • Human Extracellular Matrix is a chromatographically partially purified matrix extract derived from human placenta and comprises laminin, collagen IV, and heparin sulfate proteoglycan. (Kleinman, HK et al., US Patent 4,829,000 (1989))
  • MATRIGEL® is a soluble basement membrane extract of the Engelbreth-Holm- Swarm (EHS) tumor, gelled to form a reconstituted basement membrane. Both of these matrix products require costly biochemical isolation, purification, and synthesis techniques and thus production costs are high. In addition these matrix products are reconstituted from extracts of basement membranes and thus do not represent the structure of the basement membrane as it exists in the body. Accordingly, the study of cellular function by culturing cells in vitro is limited by the availability of cell growth substrates that present the appropriate physiological environment for the proliferation and development of the cultured cells.
  • the present invention is directed to the use of vertebrate basement tissue-derived matrices as substrates for the growth and attachment of a wide variety of cell types.
  • the extracellular matrices for use in accordance with the present invention comprise highly conserved collagens, glycoproteins, proteoglycans, and glycosaminoglycans in their natural configuration and natural concentration.
  • the extracellular matrix is derived from ovarian follicular tissue of a vertebrate species, and in particular ovarian follicular tissue of vertebrate species that lay eggs.
  • a composition comprising intact basement membrane of follicle tissue of a vertebrate is described.
  • the composition comprises basement membrane is substantially free of cells of the source vertebrate wherein the basement membrane is retained in its natural three dimensional shape.
  • Follicle basement membrane (FBM) prepared in accordance with the present invention is use to induce the proliferation and growth of cells.
  • the invention is directed to a composition and method for culturing eukaryotic cells in vitro.
  • Fig. 1 is a cross-sectional view of a follicle demonstrating the various cell layers of the follicle.
  • Figs. 2 A and 2B are photomicrographs of fibroblast cells growing on plastic (Fig. 2 A) and on FBM (Fig. 2B).
  • Figs. 3 A and 3B are photomicrographs of epithelial cells growing on plastic (Fig. 2A) and on FBM (Fig. 2B).
  • Fig. 4 is an SDS-PAGE profile (7.5% Gel, under reducing conditions) of extracts isolated from Matrigel and from FBM (left panel). The right panel includes various fractions of FBM extracted proteins: lane 1 contains a molecular weight arker; lane 2 the first fraction from batch 1; lane 3 the first fraction from batch 2; lane 4 the second fraction from batch 1; lane 5 the second fraction from batch 2.
  • Fig. 5 is a graphic representation of the effect of fraction 1 of FBM on follicle-stimulating hormone (FSH) induced steroid hormone synthesis.
  • FSH follicle-stimulating hormone
  • Fig. 6 is a graphic representation of the effect of fraction 1 of FBM on cell function in vitro.
  • Cells that are capable of producing progesterone were placed in tissue wells in which different amounts of fraction 1 had been dried. The cells were incubated for 15 hours and the amount of progesterone produced was measured. Data are mean ⁇ SEM of six incubations.
  • Figs. 7A-7D Effect of fraction 1 solubilized basal lamina protein (liquid form) on progesterone production.
  • Granulosa cells isolated from immature (SYF), first (FI), third (F3) and developing fifth, sixth and seventh (F5-7) largest preovulatory follicles were placed in 96-well plates and different amounts of fraction 1 were added as liquid. The mixture was incubated for 15 hr and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Figs. 8A-8D Effect of pre-coated fraction 1 (solid form) of solubilized basal lamina on progesterone production.
  • Granulosa cells isolated from immature (SYF), first (FI), third (F3) and developing fifth, sixth and seventh (F5-7) largest preovulatory follicles were incubated in 96-well plate that were pre-coated with different amounts of fraction 1 protein. The cells were incubated for 15 hr and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Figs. 9A-9C Dose-response effect of LH on progesterone production in the presence of the solid form of fraction 1 basal lamina.
  • Granulosa cells isolated from the first (FI), third (F3) and developing fifth, sixth and seventh (F5-7) largest preovulatory follicles were incubated in 96-well plate that were pre-coated with fraction 1 (160 ⁇ g/cm 2 ) of fluidized basal lamina proteins. The cells were incubated for 15 hr in the presence and absence of different amounts of LH and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Figs. 10A-10C Dose-response effect of the solid form of fraction 1 of basal lamina on LH-induced progesterone production.
  • Granulosa cells from the first (FI), third (F3) and fifth, sixth and seventh (F5-7) largest preovulatory follicles were incubated in 96-well plate that were pre-coated with different amounts of fraction 1 of fluidized basal lamina. The cells were incubated for 15 hr in the presence and absence of LH (5 ng/ml) and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Figs. 11 A-l IB Dose-response effect of the liquid form of fraction 1 of basal lamina on LH-induced progesterone production.
  • Granulosa cells from the first (FI), fifth, sixth and seventh (F5-7) largest preovulatory follicles were placed in 96- well plates and different amounts of fraction 1 were added as liquid. The mixture was incubated for 15 hr in the presence and absence of LH and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Figs. 12A-12C Dose-response effect of liquid form of fraction 1 of basal lamina on FSH-induced progesterone production.
  • Granulosa cells from the third (F3), developing fifth, sixth and seventh (F5-7) largest preovulatory and a pool of small yellow follicles were placed in 96-well plates and different amounts of fraction 1 were added as liquid. The mixture was incubated for 15 hr in the presence and absence of FSH (10 ng/ml) and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Figs. 13A-13C Dose-response effect of solid form of fraction 1 of basal lamina on FSH-induced progesterone production.
  • Granulosa cells from the third (F3), developing fifth, sixth and seventh (F5-7) largest preovulatory and immature (SYF) follicles were incubated in 96-well plate that were pre-coated with different amounts of fraction 1 of fluidized basal lamina. The cells were incubated for 15 hr in the presence and absence of FSH (10 ng/ml) and the progesterone content of the incubation medium was measured. Data are mean ⁇ SEM of nine incubations from three separate experiments.
  • Fig. 14 Morphometric parameters of granulosa cells incubated in wells pre-coated with fraction 1 of solubilized basal lamina. Graphic representations of the area occupied (top panels, A, B and C), perimeter (middle panels, D, E and F) and circularity (bottom panels, G, H and I) are shown. Granulosa cells isolated from mature (FI; panels A, D, G), developing (F3; panels B, E, H), and immature (SYF; panels C, F I) chicken ovarian follicles were incubated in serum-free medium 199 for 15 hr on plastic or in wells pre-coated with fraction 1 protein (5-50 ⁇ g/cm 2 ). Each point is mean ⁇ SEM of 50 or more cells.
  • Fig. 15 Morphometric parameters of granulosa cells incubated in wells to which fraction 1 of solubilized basal lamina was added as liquid. Graphic representations of the area occupied (top panels, A, B and C), perimeter (middle panels, D, E and F) and circularity (bottom panels, G, H and I) are shown.
  • Granulosa cells isolated from mature (FI; panels A, D, G), developing (F3; panels B, E, H), immature (SYF; panels C, F, I) chicken ovarian follicles were incubated in serum free medium 199 for 15 hr in the absence or presence of fraction 1 protein (50-500 ⁇ g/ml) which was added as liquid. Each point is mean ⁇ SEM of 50 or more cells.
  • the present invention is directed to an intact basement membrane prepared from vertebrate species and its use for inducing the proliferation and growth of eukaryotic cells.
  • the follicle basement membranes (FBM) of the present invention provide a three dimensional structure that enhances the proliferation and differentiation of eukaryotic cells when placed in an environment conducive to the proliferation of the cells. It has been found that FBM prepared from the follicles of vertebrate species provides a superior substrate for culturing cells in vitro.
  • cell culture substrates comprising basement membranes prepared from follicle tissue are capable of stimulating cell proliferation and inducing cell differentiation. It is also anticipated that such compositions can be used as biodegradable tissue graft constructs for implantation into vertebrate species.
  • the extracellular matrix prepared in accordance with one embodiment of the present invention comprises basement membranes isolated from the follicle tissues of egg laying vertebrate species including fish, amphibians, reptiles and birds.
  • One preferred source of follicle tissue is the membrana granulosa of avian and reptile species.
  • the membrana granulosa comprises three layers: the Perivitelline layer, the granulosa cell layer and the basal lamina, with the granulosa cell layer located between the Perivitelline layer and the basal lamina (See Fig. 1).
  • the preparation of an extracellular matrix in accordance with the present invention comprises separating the thecal layers from the granulosal layers by the method described by Gilbert et al. (J. Reprod. Fertil. 50:179-181, 1977) and then lysing the cells of the granulosa cell layer to release the Perivitelline layer and basal lamina as intact distinct layers.
  • the basal lamina of follicle tissue is not attached to connective tissues, and thus can be easily isolated from the other tissue of the membrana granulosa after destruction of the granulosa cells.
  • the preparative techniques described in the present invention provide an extracellular matrix substantially free of any cellular components. More particularly, in one embodiment the extracellular matrix of the present invention comprises intact basal lamina isolated from the membrana granulosa of avian or reptile follicle tissue, substantially free, preferably devoid, of all cells (e.g., the granulosa cells) of the avian or reptile.
  • the basement membrane compositions of the present invention are referred to herein generically as follicle basement membranes (FBM).
  • the FBM is prepared from avian ovarian follicle tissues and comprises the basal lamina of the membrana granulosa, and more particularly, the basal lamina prepared from follicle tissue comprises the lamina densa, and the lamina rara.
  • Basement membranes of tissues that are in direct contact with connective tissue may also include a third layer, the lamina reticularis. Since the basal lamina of follicle tissue is not directly attached to connective tissues, the intact basal lamina prepared from follicle tissues is typically devoid of the lamina reticularis layer.
  • basal lamina is prepared from follicle tissue harvested from avian species raised for meat or egg production, including, for example, chickens, turkeys and ostriches.
  • follicle tissue harvested from avian species raised for meat or egg production, including, for example, chickens, turkeys and ostriches.
  • Chickens provide one preferred source of follicle tissue for the isolation of intact basal lamina.
  • the structure of chicken ovarian basal lamina is granular in nature, and is similar to that described for the lamina densa layer of basement membranes of rat seminiferous tubule, vas deferens, epidermis, trachea, jejunum; monkey seminiferous tubule, mouse lens capsule, rat Reichert's membrane and rat ovarian basal lamina.
  • the preparation of follicle basement membrane from follicle tissue of a vertebrate in accordance with the present invention is carried out by removing the cellular components from the tissue.
  • the preparation of the basal lamina from follicle tissues comprises the step of removing the cellular components of the tissue and isolating the intact extracelluar matrix. Ideally the process is carried out to separate the cells from the basement membranes without damaging, or at least minimizing disruption or damage to the basement membrane.
  • the natural three dimensional structure of the original basement membrane matrix is left intact after removal of the cellular components of original follicle tissue.
  • the resulting material comprises an extracellular matrix consisting essentially of follicle basement membrane devoid of endogenous cells associated with the source vertebrate follicle tissue used to prepared the composition.
  • basal lamina is isolated from follicle tissue by contacting the follicle tissue with a cell-lysis solution for a time sufficient to lyse/release all cells from the matrix.
  • follicle tissue is treated with a buffer containing 6 M guanidine-HCl, (or 6 M guanidine thiocynate) and shaken overnight at 4°C.
  • the mixture is then centrifuged at 10,000 and the supernatant is dialyzed against 150 mM NaCl 50 mM, Tris-HCl pH 7.4 overnight at 4°C and stored frozen in the same buffer.
  • the tissue is treated with a ⁇ - mercaptoethanol (5 mM) solution containing 6 M guanidine-HCl (or guanidine thiocynate) and shaken overnight at 4°C.
  • the mixture is then centrifuged at 10,000x,g and the supernatant is dialyzed against 150 mM NaCl, 50 mM Tris-HCl pH 7.4 overnight at 4°C and stored frozen in the same buffer (solubilized form).
  • the tissue can be treated sequentially, with the guanidine and ⁇ -mercaptoethanol solutions.
  • the tissue can be treated with a solution containing 6 M guanidine-HCl and centrifuged to isolate first fraction, comprising basal lamina. The supernatant is kept as a first fraction (fraction 1) and the pellet is then treated with a ⁇ -mercaptoethanol (5 mM) solution containing 6 M guanidine-HCl (or guanidine thiocynate) to solubilize a second fraction (fraction 2) comprising basal lamina.
  • the basal lamina can also be prepared in accordance with any of the procedures described above, but substituting 8 M urea for guanidine HC1.
  • a composition comprising follicle basement membrane is prepared by suspending the follicle tissue or portions thereof in a cell-lysis solution containing one or more protease inhibitors. After contacting the follicle tissue with the cell-lysis solution for a time sufficient to lyse/release all cells from the matrix, the resulting follicle basement membrane is rinsed one or more times with saline and used immediately or optionally stored in a frozen hydrated state or a partially dehydrated state until used as described below.
  • the cell-lysis step may require several treatments with the cell-lysis solution to release substantially all cells from the basement membrane.
  • Isolation of intact follicle basement membranes in accordance with the present invention provides an acellular matrix having the three dimensional shape of the natural basal lamina.
  • the acellular basement membrane matrix comprises basal lamina delaminated from the granulosa cells of avian vertebrate follicle tissue.
  • a multi-layered FBM construct is formed by overlapping multiple sheets of follicle basement membrane and adhering the layers to each other.
  • the multi-layered FBM constructs comprising two or more sheets of FBM are preferably formed after the endogenous cells have been removed form the individual FBM sheets.
  • the individual layers can be fix to one another using standard techniques know to those skilled in the art including the use of sutures, staples and biocompatible adhesives such as collagen binder pastes.
  • the layers are fused together by compressing the overlapped regions under dehydrating conditions, optionally with the addition of heat.
  • multiple sheets of FBM are completely overlapped with each other to produce a homo-laminate structure having enhanced strength relative to a single sheet of FBM.
  • each sheet of the multi-layered construct can be partially overlapped with another sheet of FBM to create a construct having a substantially greater surface area than any one sheet used to form the construct.
  • each sheet 30-50% of each sheet is overlapped by a neighboring sheet of FBM.
  • a fluidized form of FBM is prepared by extracting bioactive components from the FBM or by comminuting and/or enzymatically digesting the prepared FBM.
  • the prepared FBM is frozen and ground to a fine powder, and the powder can be rehydrated to form a fluidized form of the FBM.
  • various components of the FBM can be extracted and solubilized to form bioactive compositions (see Example 3).
  • the viscosity of solubilized basal lamina for use in accordance with this invention can be manipulated by controlling the concentration of the basal lamina component and the degree of hydration.
  • the viscosity can be adjusted to a range of about 2 to about 300,000 cps at 25 °C.
  • the collagen component of the solubilized forms of basal lamina can be polymerized to form a solid or semi-solid matrix using standard techniques known to those skilled in the art (see for example, Kleinman et al., Biochemistry 21 : 6188-6193, 1986).
  • high viscosity formulations for example, gels
  • compositions comprising the basal lamina of the membrana granulosa provides a superior cell culture substrate for supporting growth or proliferation of eukaryotic cells in vitro. Accordingly, one embodiment of the present invention is directed to a method for supporting the growth of eukaryotic cells in vitro.
  • the method comprises the step of contacting the cells in vitro with a cell growth substrate comprising basal lamina under conditions conducive to the proliferation of said cells, wherein the basal lamina is delaminated from the membrana granulosa cells of warm-blooded vertebrate follicle tissue.
  • contacting as used herein with reference to cell culture is intended to include both direct and indirect contact, for example in fluid communication, with the basal lamina composition and the cultured cells.
  • condition conducive to eukaryotic cell growth refers to the environmental conditions, such as sterile technique, temperature and nutrient supply, that are considered optimal for eukaryotic cell growth under currently available cell culture procedures. Although optimum cell culture conditions used for culturing eukaryotic cells depend somewhat on the particular cell type, cell growth conditions are generally well known in the art.
  • differentiated cell types are still considered difficult to culture (i.e., islets of Langerhans, hepatocytes, chondrocytes, osteoblasts, etc.). It is anticipated that the follicle basement membrane compositions of the present invention can be used to stimulate proliferation of undifferentiated stems cells as well as differentiated cells such as hepatocytes and chondrocytes. Furthermore, the described cell growth composition is believed to support the growth of differentiated cells while maintaining the differentiated state of such cells.
  • basal lamina Relative to currently available cell culture substrates derived from extracellular matrices, basal lamina is quick and easy to isolate and advantageously can be used in biomedical research especially in experiments designed to assess the effects of basement membranes exactly in the form in which they exist in vivo.
  • the basal lamina can be used as a cell growth substrate in a variety of forms, including its native intact sheet-like configuration, as a solubilized solution/suspension or gel matrix, as an additive for art-recognized cell/tissue culture media, or as a coating for culture-ware to provide a more physiologically relevant substrate that supports and enhances the proliferation of cells.
  • the membrane material is preferably sterilized prior to use in cell culture applications, however nonsterile material can be used if antibiotics are included in the cell culture system.
  • the cell growth substrate of the present invention can be combined with nutrients, including minerals, amino acids, sugars, peptides, proteins, or glycoproteins that facilitate cellular proliferation, such as laminin and fibronectin and growth factors such as epidermal growth factor, platelet-derived growth factor, transforming growth factor beta, or fibroblast growth factor.
  • a cell culture substrate comprises the lamina densa delaminated from the Perivitelline layer and granulosa cell layers of follicle tissues of avian and reptilian species.
  • the basal lamina is substantially free of follicular cells of the vertebrate source of the basal lamina tissue.
  • the intact basal lamina tissue is used as the tissue culture substrate and the cells are seeded directly onto sheets of intact basal lamina under conditions conducive to eukaryotic cell proliferation.
  • the cells are seeded onto the side of the basal lamina that is in contact with the granulosa cells in vivo.
  • fluidized or powder forms of basal lamina are used to supplement standard eukaryotic culture media to enhance the standard media's capacity for sustaining and inducing the proliferation of cells cultured in vitro.
  • the present basal lamina compositions may be sterilized using conventional sterilization techniques including tanning with glutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxide treatment, propylene oxide treatment, gas plasma sterilization, gamma radiation, and peracetic acid sterilization.
  • a sterilization technique which does not significantly weaken the mechanical strength and biotropic properties of the material is preferably used. For instance, it is believed that strong gamma radiation may cause loss of strength in the basal lamina.
  • a cell culture composition for supporting growth in vitro of an eukaryotic cell population.
  • the composition comprises follicle basement membrane of a warm-blooded vertebrate in combination with nutrients, and optionally including growth factors.
  • the follicle basement membrane can be used with commercially available cell culture liquid media (both serum based and serum free), and the cells can either be in direct contact with the follicle basement membrane or simply be in fluid communication with the follicle basement membrane.
  • HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • collagenase Type IV soybean trypsin inhibitor
  • bovine serum albumin BSA, Fraction V
  • penicillin G streptomycin
  • fungizone was purchased from Sigma Chemical Co., St. Louis, MO.
  • Medium 199 (M199) containing Hank's salts was from Gibco- BRL, Grand Island, NY. Animals
  • the first and second largest (F x and F 2 ) preovulatory follicles were removed and placed in ice-cold Hank's salt solution containing NaCl 144 mM, KCl 5 mM, MgCl 2 1 mM, CaCl 2 1 mM, HEPES 10 mM, pH 7.4.
  • the thecal and granulosal layers were separated by the method described by Gilbert et al. (1977).
  • the granulosa cells were dissociated in medium 199 containing NaHCO 3 (350 mg/1), HEPES (20 mmol/1), pH 7.4, penicillin G (100,000 U/l), streptomycin (100 mg/1), fungizone (250 ⁇ g/1), collagenase (500,000 U/l) and trypsin inhibitor (200 mg/1).
  • Cell viability determined by the trypan blue exclusion method, was routinely greater than 95%.
  • the granulosal layer (membrana granulosa) was placed in a hypotonic solution containing Tris-HCl 10 mM (pH 7.4), leupeptin 0.5 mg/lit, EDTA-Na 2 1 mM, pepstatin 0.7 mg/lit, and phenylmethylsuffonyl fluoride (PMSF) 0.2 mM in a petri dish.
  • the granulosa cells, sandwiched between the basal lamina and perivitelline layer were lysed and the basal lamina and perivitelline layer were separated. The length of time required for the complete separation of the two layers (basal lamina and perivitelline layer) is dependent upon the hypotonicity of the solution.
  • Basal lamina isolated from the largest (32-35 mm in diameter) or second (27-30 mm in diameter) largest preovulatory follicles (F j and F 2 ) was spread (granulosa side up) in 35 mm Falcon or Corning culture dish and allowed to dry opened to room air in a lamina flow hood at room temperature (23 °C) for 2 hr.
  • the plates were then used following the attachment procedure or were wrapped in aluminum foil and stored in a dessicator kept at 4°C.
  • the storage of basal lamina coated culture dishes at room temperature (23 °C) or at 4°C for two years had no apparent effect on the ability of the matrix material to influence cell morphology.
  • NTH-3T3 fibroblasts NIH-3T3 cells
  • ras-transformed NIH-3T3 cells ras-3T3 cells
  • canine prostate cancer cells and human kidney carcinoma cells were kindly provided by Dr. David Waters, Department of Veterinary Clinical Sciences, Purdue University, West Lafayette.
  • Cells isolated from chick or rat embryo nervous system were kindly provided by Dr. Jim Walker, Department of Basic Medical Sciences, Purdue University, West Lafayette.
  • the cells were cultured in medium 199 containing Hank's salts, penicillin G 100.000 U/L, streptomycin 100 mg/L, fungizone 250 ⁇ g/L, Hepes 20 mM (pH 7.4), and BSA 0.1%. Unless indicated otherwise, all cells were cultured in serum- free media and without any other additive.
  • Granulosa cell layer (granulosa cells, basal lamina, perivitelline membrane) or isolated basal lamina or perivitelline membrane was fixed in 3% glutaraldehyde in 0.15 M Millonig's phosphate buffer pH 7.2 for 24 hr. The layer was then rinsed in phosphate buffer and postfixed in 1 % osmium tetroxide-1.5% potassium ferrocyanide at 4 °C for 1.5 hr. Tissue was then rinsed in buffer, dehydrated through a graded ethanol series, rinsed 2X in propylene oxide and infiltrated with epoxy resin
  • Granulosal layer (granulosa cells, basal lamina, perivitelline membrane) or isolated basal lamina or perivitelline membrane was fixed in 3% glutaraldehyde in 0.15 M Millonig's phosphate buffer pH 7.2 for 24 hr. The tissue was then rinsed in phosphate buffer and postfixed in 1% osmium tetroxide-1.5% potassium ferrocyanide at 4°C for 1.5 hr. Tissue was then rinsed in buffer, dehydrated through a graded ethanol series, and then a graded freon 113 series. The granulosa cell layer or the individual components were then picked up from a petri dish containing freon 113 with a glass microscope coverslip and air dried. The coverslips were glued to aluminum stubs with silver paint, sputter coated with gold, and examined in an ISI 100 A scanning electron microscope.
  • the perivitelline layer in the preovulatory follicle contains a network of thick fibers that appear as electron dense elongated rods when examined by transmission electron microscopy
  • the basal lamina appears to be made up of a network of fuzzy strands that are irregularly arranged and are separated by spaces Similar structures (fuzzy strands) or arrangements have been reported in basement membranes and are referred to as cords in the scientific literature (see Inoue and Leblond, 1988)
  • the structure of both basal lamina and perivitelline layer appeared to be unaltered following isolation by the procedure described in materials and methods
  • basal lamina The effect of basal lamina on cell morphology was assessed by observing the morphology of cells cultured in vitro on basal lamina attached to the bottom of 35 mm culture dishes Cells were plated on Falcon or Corning 35 mm dishes that did not contain basal lamina served as controls
  • NIH-3T3 cells sparsely seeded fibroblasts (NIH-3T3 cells) were attached and exhibiting a flattened shape on the plastic control dishes in serum free medium 199
  • the cells extended cellular projections and there was little or no cluster formation (aggregation)
  • the NIH-3T3 cells that attached to basal lamina were rounded, some were oval in shape (much less elongated) with few or no, cellular projections
  • the NIH-3T3 cells that attached to plastic in basal lamina-containing dishes were less elongated
  • ras-3T3 cells sparsely seeded ras- transformed fibroblasts (ras-3T3 cells) were elongated, and exhibited a spindly and flattened shape on the plastic control dishes
  • well defined long cellular projections were observed in control dishes and there was little or no cluster formation (aggregation)
  • the ras-3T3 cells that attached to basal lamina appeared oval in shape (much less elongated) with few or no cytoplasmic extensions Some of the cells were spherical in shape
  • the ras-3T3 cells tended to aggregate, others formed overlapping clusters on FBM Some of them appear to have penetrated and become situated beneath the FBM
  • the ras-3T3 cells that attached to plastic in basal lamina-containing dishes were less elongated
  • solubilized avian basal lamina was resolved by SDS-PAGE on gradient gels (4 - 20%) as well as on high (12.5%), medium (7.5%) and low (5%) polyacrylamide containing gels and stained with coomassie blue or silver stain Proteins in avian basal lamina solubilized in one step and avian basal lamina solubilized in two-steps (first- and second-fractions) were resolved
  • the first-fraction contained more bands (with coomassie blue staining) than the second one In the first-fraction, the range of mol. wt of the proteins is from 10 kDa to 1000 kDa
  • the range of mol wt of the proteins in the second-fraction is from 40 kDa to 1000 kDa; most of them are between 40 kDa and 130 kDa
  • Some proteins present in the second-fraction with mol wt between 130 kDa and 200 kDa were absent from the first-fraction Specifically, a 130 kDa protein, the major protein in the second-fraction is not present in the first-fraction It is noteworthy that the proteins in the second-fraction stain well with coomasie blue but very poorly with silver stain
  • Fibroblasts (NIH-3T3 cells) and ras-transformed fibroblasts (ras-3T3 cells) were flat, elongated with long extensions on plastic (Fig. 3A). However, those attached to follicle basement membrane appeared oval (some were rounded) in shape with few extensions (Fig. 3B). Human vascular endothelial cells and human renal cancer cells (A4982) were also spread and flattened on plastic but became rounded and sphe ⁇ cal on follicle basement membrane (data not shown) The storage of follicle basement membrane at 40 °C had no apparent effect on its ability to influence cell shape (data not shown)
  • the follicle basement membrane was solubilized in one step with a mixture of guanidine-HCl and ⁇ -mercaptoethanol (total extract, Fig 4A) and designated FBM-Gel It was also solubilized in two steps with guamdine-HCI alone (fraction 1) and subsequently with a mixture of guamdine-HCI and ⁇ -mercaptoethanol (fraction 2)
  • the solubilized proteins are dialyzed and stored in physiological salt solution buffered with 50 mM T ⁇ s-HCI (pH 7 4)
  • Solubilized FBM was prepared in accordance with Example 3 Solubilized first-fraction was diluted with deiomzed water and different amounts were transferred to tissue culture wells The protein was allowed to dry completely in tissue culture wells at room temperature The wells contained 2, 10 or 20 ⁇ g fraction 1 protein (that is 1, 5 or 10 ⁇ g fraction 1 per CM 2 ) Control wells received deiomzed water alone When dried, a clear layer of material could be seen at the bottom of the wells In wells that contained greater amounts of fraction 1 e g 30 ⁇ g/CM 2 or more, the dried material is slightly cloudy in appearance However, this did not interfere with studies under the light microscope
  • Epithelial cells were then seeded in the wells and incubated for 15 hr at 37 °C
  • the cells in control wells were flattened, spread with cytoplasmic extensions
  • the cells in wells that contained fraction 1 were rounded in shape
  • the degree of change in shape was dependent on the amount of fraction 1 in the wells
  • Significant change in cell shape was observed in wells containing as low 1 ⁇ g/cm 2 fraction 1
  • the effect of the solubilized basement membrane (fraction 1) on cell shape was similar to that observed for intact follicle-basement membrane Therefore, the follicle basement membrane can be solubilized without any apparent loss of its properties
  • the FBM-Gel allows for the preparation of low cost extracellular matrix that is fully competitive with the available industry standard (Matrigel)
  • Extracellular Matrix Proteins Fibronectin, tenascin, osteonectin (SPARC or BM-40), type IV collagen, entactin/nidogen, heparan sulfate proteoglycan (HSPG) laminin Basement membranes (basal lamina) have different proportions of collagen as well as non-collagenous extracellular matrix proteins such as, laminin and heparan sulfate proteoglycans (HSPG) Follicle basement membrane contains fibronectin which is absent from Matrigel/ECM-gel
  • Acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor- (TGF- ), insulin-like growth factor-I (IGF-1), insulin-like growth factor- II (IGF-II), platelet-derived growth factor-AA (PDGF-AA), transforming growth factor- ⁇ (TGF- ⁇ l, -2, -3, -5).
  • aFGF Acidic fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • TGF- insulin-like growth factor-I
  • IGF-II insulin-like growth factor- II
  • PDGF-AA platelet-derived growth factor-AA
  • TGF- ⁇ l transforming growth factor- ⁇ l, -2, -3, -5.
  • Insulin-like Growth Factor Binding Proteins Insulin-like growth factor binding proteins (IGFBPs) IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6 and IGFBP-7
  • Cytokmes/Hematopoietic Factors Interferon- ⁇ (IFN- ⁇ ), interleukin-3 (IL-3), granulocyte macrophage-colony stimulating factor (GM-CSF)
  • IFN- ⁇ Interferon- ⁇
  • IL-3 interleukin-3
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • MMPs Matrix Metalloproteinases
  • Tissue Inhibitors of Matrix Metalloproteinases TIMP-1, TLMP-2, TIMP-3, TIMP-4
  • Plasminogen Activators and their Inhibitors Tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1)
  • t-PA Tissue-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • PAI-1 type 1 plasminogen activator inhibitor
  • Fraction 1 of the solubilized basal lamina was diluted with deiomzed water or modified Hanks salt solution or medium 199 Aliquots of 100-200 ⁇ l containing 10-160 ⁇ g of proteins were transferred into 96-well or 24-well Falcon culture dishes (Fisher Scientific) and allowed to dry under tissue culture hood These are designated pre-coated wells (solid form of solubilized basal lamina) Some wells received vehicle only and served as controls Culture wells that received Hanks salt solution or medium 199 were rinsed two times with deiomzed water prior to the incubation of cells Tissue isolation, solubilization, dialysis and preparation of culture dishes were carried out under sterile conditions
  • Progesterone is the primary steroid hormone produced by chicken granulosa cells and its production varies with the state of cell differentiation (state of follicular development)
  • the effects of fraction 1 of solubilized basal lamina on progesterone synthesis in chicken granulosa cells were examined in (1) experiments in which different amounts of fraction 1 were added as liquid (liquid form) to the incubation medium or (2) in experiments in which granulosa cells were incubated in culture wells in which fraction 1 had been dried (solid form)
  • Fraction 1 of solubilized basal lamina, added as liquid to the incubation medium caused increase in progesterone production in a concentration dependent manner in granulosa cells obtained from mature (FI), developing (F3, F5-7) and immature (SYF) follicles (Figs 7A-D)
  • the slope of the regression line was greater for SYF and F5-7 than for F3 or FI granulosa cells
  • a unit concentration of fraction 1 caused a greater increase in the quantity of progesterone produced by less differentiated SYF granulosa cells than the amount of progesterone produced by differentiating F3 or differentiated FI granulosa cells
  • the stimulatory effect of fraction 1, added as liquid on progesterone production was least in wells that contained FI cells (Figs 7A-D)
  • fraction 1 protein did not suppress the stimulatory action of LH (Figs 1 1 A and 1 IB) irrespective of the state of granulosa cell differentiation
  • the stimulatory effect of LH on progesterone production in both differentiated (FI) and differentiating (F5-7) granulosa cells was further enhanced (albeit marginally) by the liquid form of fraction 1 (Figs 1 1 A and 1 IB)
  • the amount of progesterone production caused by LH was greater in granulosa cells from mature (FI) follicles than in granulosa cells in developing (F3 or F5-7) cells (Figs 9, 10 & 1 1) Effect of Solubilized Basal Lamina on FSH-induced Progesterone Production In Vitro
  • Fig 14 Dried fraction 1 reduced the mean area occupied by granulosa cells at all stages of differentiation (Fig 14A-C) Similarly, the perimeter of cells incubated in fraction 1 pre-coated wells was less thanthat of cells incubated on plastic (Fig 14D-F) Moreover, the cells incubated on plastic were more irregular than those incubated on fraction 1 (Fig 14G-I) As such, in wells pre-coated with fraction 1 of solubilized basal lamina, granulosa cells assumed a morphology that approximated the shape of chicken granulosa cells in vivo (in intact membrane granulosa) Granulosa cells incubated on dried fraction 1 formed clusters(data not shown) similar to what has been observed
  • Fig. 15 shows the morphometric parameters of granulosa cells incubated in wells to which fraction 1 was added as liquid.
  • FI differentiated
  • F3 differentiating
  • SYF undifferentiated
  • the structure of pure and intact basal lamina isolated from hen ovarian follicle is similar to that observed for basal lamina in the intact follicle. It influenced the shape of granulosa cells, the cells that it is in association with in vitro. Both the intact basal lamina and its fluidized form can be stored (preferrably at 4°C and in a dehydrated state) for 1-2 years without losing their ability to influence cell shape.
  • One advantage of the utility of intact basal lamina is that it could provide data on in vivo behavior (responses); it can be used for the culture of cells in experiments designed to examine the influence of the basement membrane microenvironment on cell structure and function. For example, this natural basal lamina can be used to test in vitro, the potential of tumor cells to metastasize.

Abstract

L'invention porte sur une préparation de membranes basales de follicules pouvant servir de substrat pour la culture de cellules destinées à proliférer in vitro.
PCT/US1998/027289 1997-12-23 1998-12-22 Materiel biologique derivant de membranes basale de follicules WO1999032607A1 (fr)

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AU20907/99A AU2090799A (en) 1997-12-23 1998-12-22 Biomaterial derived from follicle basement membranes
EP98965443A EP1042453A4 (fr) 1997-12-23 1998-12-22 Materiel biologique derivant de membranes basale de follicules
US09/582,179 US6485969B1 (en) 1997-12-23 1998-12-22 Biomaterial derived from follicle basement membranes

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US20040043006A1 (en) * 2002-08-27 2004-03-04 Badylak Stephen F. Tissue regenerative composition
US6576265B1 (en) 1999-12-22 2003-06-10 Acell, Inc. Tissue regenerative composition, method of making, and method of use thereof
US20040175366A1 (en) * 2003-03-07 2004-09-09 Acell, Inc. Scaffold for cell growth and differentiation
US20040176855A1 (en) * 2003-03-07 2004-09-09 Acell, Inc. Decellularized liver for repair of tissue and treatment of organ deficiency
WO2005002601A1 (fr) * 2003-06-25 2005-01-13 Badylak Stephen F Compositions matricielles conditionnees pour la restauration d'un tissu
ATE469209T1 (de) * 2005-05-30 2010-06-15 Commw Scient Ind Res Org Herstellung und verwendung von basalmembranpartikeln
US20070190165A1 (en) * 2005-10-21 2007-08-16 Brey Eric M Tissue-specific basement membrane gels
US8652500B2 (en) 2009-07-22 2014-02-18 Acell, Inc. Particulate tissue graft with components of differing density and methods of making and using the same
US8298586B2 (en) * 2009-07-22 2012-10-30 Acell Inc Variable density tissue graft composition
US10617790B2 (en) * 2013-01-09 2020-04-14 Ise Professional Testing & Consulting Services, Inc. Decellularized biomaterial from non-mammalian tissue
US9238090B1 (en) 2014-12-24 2016-01-19 Fettech, Llc Tissue-based compositions

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US4921808A (en) * 1986-06-25 1990-05-01 The Albany Medical College Of Union University Method for determining follicle stimulating hormone
US4902508A (en) 1988-07-11 1990-02-20 Purdue Research Foundation Tissue graft composition
US5821121A (en) * 1991-06-24 1998-10-13 Pacific Biomedical Research, Inc. Hormone-secreting cells maintained in long-term culture
US5281422A (en) 1991-09-24 1994-01-25 Purdue Research Foundation Graft for promoting autogenous tissue growth
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